Longitudinal metabolomics of human plasma reveal metabolic dynamics and predictive markers of antituberculosis drug-induced liver injury

Tuberculosis (TB) remains the second leading cause of death from a single infectious agent and long-term medication could lead to antituberculosis drug-induced liver injury (ATB-DILI). We established a prospective longitudinal cohort of ATB-DILI with multiple timepoint blood sampling and used untargeted metabolomics to analyze the metabolic profiles of 107 plasma samples from healthy controls and newly diagnosed TB patients who either developed ATB-DILI within 2 months of anti-TB treatment (ATB-DILI subjects) or completed their treatment without any adverse drug reaction (ATB-Ctrl subjects). The untargeted metabolome revealed that 77 metabolites (of 895 total) were significantly changed with ATB-DILI progression. Among them, levels of multiple fatty acids and bile acids significantly increased over time in ATB-DILI subjects. Meanwhile, metabolites of the same class were highly correlated with each other and pathway analysis indicated both fatty acids metabolism and bile acids metabolism were up-regulated with ATB-DILI progression. The targeted metabolome further validated that 5 fatty acids had prediction capability at the early stage of the disease and 6 bile acids had a better diagnostic performance when ATB-DILI occurred. These findings provide evidence indicating that fatty acids metabolism and bile acids metabolism play a vital role during ATB-DILI progression. Our report adds a dynamic perspective better to understand the pathological process of ATB-DILI in clinical settings. Supplementary Information The online version contains supplementary material available at 10.1186/s12931-024-02837-8.

Then, the mixtures were vigorously shaken for 2 min followed by centrifugation, thus proteins were precipitated and small molecules were released.The resulting supernatants were divided into five fractions: two for analysis by two separate reverse phase (RP) ultraperformance liquid chromatography/tandem mass spectrometry (UPLC-MS/MS) methods with positive ion mode electrospray ionization (ESI), one for analysis by RP/UPLC-MS/MS with negative ion mode ESI, one for analysis by hydrophilic-interaction chromatography (HILIC) UPLC-MS/MS with negative ion mode ESI, and one was reserved for backup.Samples were placed briefly on a TurboVap® (Zymark) to remove the organic solvent.The sample extracts were stored overnight under nitrogen before preparation for analysis.
All methods utilized a Waters ACQUITY ultra-performance liquid chromatography and a Thermo Scientific Q-Exactive high resolution/accurate mass spectrometer interfaced with a heated electrospray ionization (HESI-II) source and Orbitrap mass analyzer operated at 35,000 mass resolution.The sample extract was dried then reconstituted in solvents compatible to each of the four methods.Each reconstitution solvent contained a series of standards at fixed concentrations to ensure injection and chromatographic consistency.One aliquot was analyzed under positive electron spray ionization (ESI), chromatographically optimized for more hydrophilic compounds.In this method, the extract was gradient eluted from a C18 column (Waters UPLC BEH C18-2.1x100mm, 1.7 µm) using water and methanol, containing 0.05% perfluoropentanoic acid (PFPA) and 0.1% formic acid.Another aliquot was also analyzed under ESI positive mode, and it was chromatographically optimized for more hydrophobic compounds.In this method, the extract was gradient eluted from the same afore mentioned C18 column using methanol, acetonitrile, water, 0.05% PFPA and 0.01% formic acid and was operated at an overall higher organic content.The third aliquot was analyzed using negative ESI mode optimized conditions using a separate dedicated C18 column.The basic extracts were gradient eluted from the column using methanol and water, however with 6.5 mM Ammonium Bicarbonate at pH 8.The fourth aliquot was analyzed using negative ESI mode following elution from a HILIC column (Waters UPLC BEH Amide 2.1x150 mm, 1.7 µm) using a gradient consisting of water and acetonitrile with 10 mM Ammonium Formate, pH 10.8.The MS analysis alternated between MS and datadependent MSn scans using dynamic exclusion.The scan range varied slightly between methods but covered 70-1000 m/z.Several types of controls were analyzed in concert with the experimental samples: a pooled matrix sample generated by taking a small volume of each experimental sample served as a technical replicate throughout the data set; extracted water samples served as process blanks; and a cocktail of quality control (QC) standards that were carefully chosen not to interfere with the measurement of endogenous compounds were spiked into every analyzed sample, allowed instrument performance monitoring and aided chromatographic alignment.Instrument variability was determined by calculating the median relative standard deviation (RSD) for the standards that were added to each sample prior to injection into the mass spectrometers.The overall process variability was determined by calculating the median RSD for all endogenous metabolites present in 100% of the pooled matrix samples.Experimental samples were randomized across the platform run with QC samples spaced evenly among the injections.
Raw data was extracted, peak-identified and QC processed using Discovery HD4TM's hardware and software.Compounds were identified by comparison to library entries of purified standards or recurrent unknown entities.Discovery HD4 TM maintains a library based on authenticated standards that contains the retention time/index (RI), mass to charge ratio (m/z), and chromatographic data (including MS/MS spectral data) on all molecules present in the library.Biochemical identifications are based on three criteria: retention index within 200ms of the proposed identification, accurate mass match to the library within (-5ppm, 5ppm), and the MS/MS forward and reverse scores between the experimental data and authentic standards.The MS/MS scores are based on a comparison of the ions present in the experimental spectrum to the ions present in the library spectrum.All identification results generated by software were manually double-checked for the accuracy.The dataset was normalized with median and missing values were imputed with minimum values.

Quantitative analysis of fatty acids
FAs were quantitatively measured according to the commercial kit instruction (Qlife, China).An aliquot of 50 L plasma sample was mixed with 450 L working solution containing internal standards.The mixture was vigorously shaken for 5 min, then centrifuged at 12000 rpm at 4℃ for 5 min.The supernatant was transferred to vials for UPLC-MS/MS analysis.Simultaneously, a series of standard calibration samples and QC samples were pretreated for calibration curve and quality assurance.
The UPLC-MS/MS system consisted of a Waters AUQUITY UPLC and a Waters XEVO TQS instrument equipped with an ESI source (Waters, the United States of America).All chromatographic separation solution was prepared according to kit instruction.Phase A was diluted 100 times with water and phase B was diluted 100 times with the mixture of acetonitrile and isopropanol at 1:1.The extracts were gradient-eluted from C18 column (Waters UPLC BEH C18-2.1x100mm, 1.7 µm).The flow rate was 0.3 mL/min with the following mobile phase gradient: 0-2.7min (73%B), 2.7-4.5min(98%B), 4.5-6.5min(73%B).The column was maintained at 40℃ and the injection volume for all samples was 1L.The extracts were analyzed under negative ESI mode and the data were collected with multiple reaction monitor (MRM).

Quantitative analysis of bile acids
BAs were quantified using commercial kit-based method and in-house established method.
Commercial kit-based method: A total of 4 BAs were quantitatively measured according to the commercial kit instruction (Qlife, China).An aliquot of 50 L plasma sample was mixed with 200L working solution containing internal standards.The mixture was vigorously shaken for 5 min, then centrifuged at 12000rpm at 4℃ for 5 min.The supernatant was transferred to a 96-well plate for UPLC-MS/MS analysis.
Simultaneously, a series of standard calibration samples and QC samples were pretreated for calibration curve and quality assurance.